Determining the type and relative proportion of an individuals' cell surface or membrane-associated proteins is medically useful because over-expression, under-expression or complete lack of certain receptors or transmembrane channels frequently is indicative of disease or state of disease. If certain receptors are over-expressed, certain drugs may cause adverse events or toxicity. Conversely, if certain receptors are under-expressed or completely absent, certain drugs may not be effective, and signal transduction may not occur.
Human leukocyte antigens (HLA) represent a class of cell surface proteins (also referred to herein as transplantation antigens), whose great variability from one individual to another forms the molecular basis for the immune system's ability to distinguish “self” from “non-self” cells and tissues. Individuals sensitized to HLA, for example in the course of pregnancy, or as a result of blood transfusion or organ transplantation, develop allo-antibodies, also referred to as “panel-active antibodies” (PRA). The presence in a prospective transplant recipient of antibodies against donor HLA alleles, also known as a “donor-specific cross-match,” is predictive of a high risk of graft rejection. It is standard practice in transplantation medicine to test all potential recipients against a panel of HLA antigens selected to represent a human population and to determine the percentage of HLA alleles against which the recipient's serum is reactive. In this “panel-reactive antibody” (PRA) testing, serum reactivity against a high percentage of HLA alleles present in a normal human population is predictive of a high risk of graft rejection. To minimize the risk of an adverse immune response leading to rejection of grafts of tissue, organ, bone marrow or transfused blood, recipients are tested to ascertain their spectrum of allo-antibodies directed against the donor's HLA spectrum (or, in the case of blood transfusion, blood group antigens).
Methods known in the art for HLA testing include the complement-dependant lymphocytotoxicity test in which serum from a recipient is incubated with donor or panel lymphocytes (such lymphocytes being representative of a spectrum of HLA in the general population) followed by incubation with complement. The level of cytotoxicity is then estimated by discriminating between dead and viable cells using a dye. This method is labor intensive, requires viable cells, may be nonspecific and requires a subjective evaluation of the cells.
Pouletty et al., U.S. Pat. No. 5,223,397, disclose methods for testing HLA compatibility between a donor and a recipient comprising the steps of adding blood from the donor to a substrate having anti-HLA antibodies bound thereto, and incubating for sufficient time for soluble HLA antigens present in the blood to bind to the antibodies or ligand. Blood from the recipient is then added to the solid substrate, whereby any antibody specific for any HLA antigens bound to the solid substrate may become bound. The detection of an absence of antibodies from the recipient's blood to the HLA antigen is indicative of a suitable cross-match.
Zaer et al., “Antibody screening by enzyme-linked immunosorbent assay using pooled soluble HLA in renal transplant candidates,” Transplantation 63: 48-51 (1997) discloses use of an ELISA using HLA class I molecules purified from pooled platelets to detect anti-HLA antibodies. In patients found to not be sensitized, the incidence of false-positive results was less for ELISA testing than for panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. In such cases, the incidence of false-negative results was higher for ELISA testing than for panel studies.
Flow cytometry assay methods have been used for analysis of membrane antigens and antibodies thereto. Wilson et al., “A new microsphere-based immunofluorescence assay for antibodies to membrane-associated antigens,” J. Immunol. Methods 107: 231-237 (1988) disclose the use of polyacrylamide microspheres coupled with cell membrane proteins in immunofluorescence assays for antibodies to membrane-associated antigens. The method is said to make possible the rapid flow cytometric analysis of plasma membrane antigens from cell populations that would otherwise be unsuitable for use in flow cytometry.
Scillian et al., “Early detection of antibodies against rDNA-produced HIV proteins with a flow cytometric assay,” Blood 73: 2041-2048 (1989) disclose the use of immunoreactive beads in flow cytometric assays for detection of antibodies to HIV. Frengen et al., Clin. Chem. 40/3: 420-425 (1993) disclose the use of flow cytometry for particle-based immunoassays of alpha-fetoprotein (AFP). This reference further reports the ability of serum factors to cross-link labeled mouse monoclonal antibodies of irrelevant specificity to different particle types coated with various immunoglobulins.
Flow cytometry methods using lymphocytes encounter difficulties arising from the activity of auto-antibodies, as reported in Shroyer et al., Transplantation 59:626-630. Moreover, when using flow cytometry with lymphocytes, use of ten or more different lymphocytes tends to produce confusing signals. As a consequence, studies using lymphocytes have been limited to presenting a small panel of HLA antigens that do not adequately reflect the distribution of HLA antigens in a normal human population.
Sumitran-Karuppan et al., “The use of magnetic beads coated with soluble HLA class I or class II proteins in antibody screening and for specificity,” Transplantation 61: 1539-1545 (1996) disclose the use of magnetic beads which use an anti-HLA capture antibody to immobilize a variety of soluble HLA antigens pooled from 80 to 100 individuals on each bead. The beads can then be directly added to patient serum for efficient absorption of HLA antibodies. The reference discloses visualization of antibody binding to the antigen-coated beads using flow cytometry and suggests that this will allow testing for antibody specificity for cross-matching purposes and for the screening of panel-reactive antibodies. The methods of Sumitran-Karuppan are limited, however, because the pooling of antigens causes sensitivity to certain rare HLA antigens. Moreover, the method is not capable of quantifying the relative amounts of PRA.
Flow cytometry analysis is performed as a separate analytical step after completion of the assay for profiling of allo-antibodies. What is needed is an analysis that integrates the assay with instant subsequent read-out, thereby facilitating greater convenience, ease-of-use and high sample throughput, hence enhancing productivity. The method should provide a universal platfonm for the quantitative analysis of proteins, nucleic acids and cells.